ABSTRACT
Introduction: High-alkalinity water is a serious health hazard for fish and can cause oxidative stress and metabolic dysregulation in fish livers. However, the molecular mechanism of liver damage caused by high alkalinity in fish is unclear. Methods: In this study, 180 carp were randomly divided into a control (C) group and a high-alkalinity (A25) group and were cultured for 56 days. High-alkalinity-induced liver injury was analysed using histopathological, whole-transcriptome, and metabolomic analyses. Results: Many autophagic bodies and abundant mitochondrial membrane damage were observed in the A25 group. High alkalinity decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity and the total antioxidant capacity (T-AOC) and increased the malondialdehyde (MDA) content in liver tissues, causing oxidative stress in the liver. Transcriptome analysis revealed 61 differentially expressed microRNAs (miRNAs) and 4008 differentially expressed mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that mammalian target of rapamycin (mTOR), forkhead box O (FoxO), mitogen-activated protein kinase (MAPK), and the autophagy signalling pathway were the molecular mechanisms involved. High alkalinity causes oxidative stress and autophagy and results in autophagic damage in the liver. Bioinformatic predictions indicated that Unc-51 Like Kinase 2 (ULK2) was a potential target gene for miR-140-5p, demonstrating that high alkalinity triggered autophagy through the miR-140-5p-ULK2 axis. Metabolomic analysis revealed that the concentrations of cortisol 21-sulfate and beta-aminopropionitrile were significantly increased, while those of creatine and uracil were significantly decreased. Discussion: The effects of high alkalinity on oxidative stress and autophagy injury in the liver were analysed using whole-transcriptome miRNA-mRNA networks and metabolomics approaches. Our study provides new insights into liver injury caused by highly alkaline water.
Subject(s)
Autophagy , Liver , Metabolome , Oxidative Stress , Transcriptome , Animals , Liver/metabolism , Liver/pathology , Gene Expression Profiling , Alkalies/toxicity , Alkalies/adverse effects , MicroRNAs/genetics , Metabolomics , Fish Diseases/metabolismABSTRACT
Corneal alkali burns often occur in industrial production and daily life, combined with infection, and may cause severe eye disease. Oxidative stress and neovascularization (NV) are important factors leading to a poor prognosis. URP20 is an antimicrobial peptide that has been proven to treat bacterial keratitis in rats through antibacterial and anti-NV effects. Therefore, in this study, the protective effect and influence mechanism of URP20 were explored in a rat model of alkali burn together with pathogenic bacteria (Staphylococcus aureus and Escherichia coli) infection. In addition, human umbilical vein endothelial cells (HUVECs) and human corneal epithelial cells (HCECs) were selected to verify the effects of URP20 on vascularization and oxidative stress. The results showed that URP20 treatment could protect corneal tissue, reduce corneal turbidity, and reduce the NV pathological score. Furthermore, URP20 significantly inhibited the expression of the vascularization marker proteins VEGFR2 and CD31. URP20 also reduced the migration ability of HUVECs. In terms of oxidative stress, URP20 significantly upregulated SOD and GSH contents in corneal tissue and HCECs (treated with 200 µM H2O2) and promoted the expression of the antioxidant protein Nrf2/HO-1. At the same time, MDA and ROS levels were also inhibited. In conclusion, URP20 could improve corneal injury combined with bacterial infection in rats caused by alkali burns through antibacterial, anti-NV, and antioxidant activities.
Subject(s)
Bacterial Infections , Burns, Chemical , Corneal Injuries , Corneal Neovascularization , Eye Burns , Rats , Humans , Animals , Burns, Chemical/complications , Burns, Chemical/drug therapy , Burns, Chemical/metabolism , Corneal Neovascularization/metabolism , Antioxidants/pharmacology , Antioxidants/therapeutic use , Hydrogen Peroxide/pharmacology , Neovascularization, Pathologic/metabolism , Corneal Injuries/drug therapy , Human Umbilical Vein Endothelial Cells , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/pharmacology , Eye Burns/chemically induced , Eye Burns/drug therapy , Eye Burns/pathology , Disease Models, Animal , Alkalies/toxicityABSTRACT
As one of the major abiotic stresses restricting the development of global agriculture, saline-alkali stress causes osmotic stress, ion poisoning, ROS damage and high pH damage, which seriously restrict sustainable development of fruit industry. Therefore, it is essential to develop and cultivate saline-alkali-resistant apple rootstocks to improve the yield and quality of apples in China. Based on transcriptome data, MhANR (LOC114827797), which is significantly induced by saline-alkali stress, was cloned from Malus halliana. The physicochemical properties, evolutionary relationships and cis-acting elements were analysed. Subsequently, the tolerance of MhANR overexpression in Arabidopsis thaliana, tobacco, and apple calli to saline-alkali stress was verified through genetic transformation. Transgenic plants contained less Chl a, Chl b and proline, SOD, POD and CAT activity, and higher relative electrical conductivity (REC) compared to WT plants under saline-alkali stress. In addition, expression of saline-alkali stress-related genes in overexpressed apple calli were also lower than in WT calli, including the antioxidant genes (MhSOD and MhCAT^), the Na+ transporter genes (MhCAX5, MhCAX5, MhSOS1, MhALT1), and the H+ -ATPase genes (MhAHA2 and MhAHA8), while expression of the K+ transporter genes (MhSKOR and MhNHX4) were higher. Expression of MhANR reduced tolerance of A. thaliana, tobacco, and apple calli to saline-alkali stress by regulating osmoregulatory substances, chlorophyll content, antioxidant enzyme activity, and expression of saline-alkali stress-related genes. This research provides a theoretical basis for cultivating apple rootstocks with effective saline-alkali stress tolerance.
Subject(s)
Arabidopsis , Malus , Malus/metabolism , Antioxidants/metabolism , Alkalies/toxicity , Alkalies/metabolism , Stress, Physiological/genetics , Chlorophyll/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , Plants, Genetically Modified/metabolism , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.
Subject(s)
Burns, Chemical , Corneal Injuries , Corneal Neovascularization , Eye Burns , Mice , Animals , Matrix Metalloproteinase 2/metabolism , Burns, Chemical/metabolism , Corneal Neovascularization/metabolism , Tumor Necrosis Factor-alpha/metabolism , Sirolimus/pharmacology , Sirolimus/therapeutic use , Alkalies/toxicity , Cornea/metabolism , Neovascularization, Pathologic/metabolism , Corneal Injuries/metabolism , Inflammation/metabolism , Cytokines/metabolism , Eye Burns/chemically induced , Eye Burns/drug therapy , Eye Burns/pathology , Disease Models, AnimalABSTRACT
PURPOSE: To determine pathological changes of meibomian glands (MGs) after transient exposure of the rat eyelid margin to alkali solution. METHODS: Filter paper infiltrated with 1 N sodium hydroxide solution was applied to the eyelid margin of Sprague-Dawley rats for 30 s under general anesthesia, without touching the conjunctiva, after which the ocular surface and eyelid margin were examined by slit-lamp microscopy. In vivo confocal microscopy and stereomicroscopy were subsequently applied to observe MG morphology on day 5, day 10 and day 30 post alkali injury. Eyelid cross-sections were processed for H&E staining, Oil red O staining and immunofluorescent staining. RESULTS: After alkali injury, there was marked plugging of MG orifices, telangiectasia and hypertrophy of the eyelid margin, while corneal epithelium was intact at post-injury days 5 and 10. However, 30 days after alkali injury, mild corneal epithelial damage was observed. Degeneration of MG acini was observed at days 5 and became aggravated at days 10 and 30, along with MG duct dilation and acini loss. Oil red O staining showed lipid accumulation in the dilated duct. Inflammatory cell infiltration and the presence of apoptotic cells was seen in the MG loci 5 days post injury, but diminished at days 10 and 30. Cytokeratin 10 expression was increased in dilated duct, while cytokeratin 14, PPAR-γ, Ki67 and LRIG1 expression were decreased in the acini of injured loci. CONCLUSIONS: Transitory alkali exposure of the rat eyelid margin obstructs the MG orifice and induces pathological changes of MG dysfunction.
Subject(s)
Corneal Injuries , Eyelid Diseases , Meibomian Gland Dysfunction , Animals , Rats , Meibomian Glands/metabolism , Meibomian Gland Dysfunction/metabolism , Eyelid Diseases/metabolism , Rats, Sprague-Dawley , Corneal Injuries/metabolism , Alkalies/toxicity , Alkalies/metabolism , Tears/metabolismABSTRACT
Corneal alkali burn (AB) is a blindness-causing ocular trauma commonly seen in clinics. An excessive inflammatory reaction and stromal collagen degradation contribute to corneal pathological damage. Luteolin (LUT) has been studied for its anti-inflammatory effects. In this study, the effect of LUT on cornea stromal collagen degradation and inflammatory damage in rats with corneal alkali burn was investigated. After corneal alkali burn, rats were randomly assigned to the AB group and AB + LUT group and received an injection of saline and LUT (200 mg/kg) once daily. Subsequently, corneal opacity, epithelial defects, inflammation and neovascularization (NV) were observed and recorded on Days 1, 2, 3, 7 and 14 post-injury. The concentration of LUT in ocular surface tissues and anterior chamber, as well as the levels of collagen degradation, inflammatory cytokines, matrix metalloproteinases (MMPs) and their activity in the cornea were detected. Human corneal fibroblasts (HCFs) were co-cultured with interleukin (IL)-1ß and LUT. Cell proliferation and apoptosis were assessed by CCK-8 assay and flow cytometry respectively. Measurement of hydroxyproline (HYP) in culture supernatants was used to quantify the amount of collagen degradation. Plasmin activity was also assessed. ELISA or real-time PCR was used to detect the production of matrix metalloproteinases (MMPs), IL-8, IL-6 and monocyte chemotactic protein (MCP)-1. Furthermore, the immunoblot method was used to assess the phosphorylation of mitogen-activated protein kinases (MAPKs), transforming growth factor-ß-activated kinase (TAK)-1, activator protein-1 (AP-1) and inhibitory protein IκB-α. At last, immunofluorescence staining helped to develop nuclear factor (NF)-κB. LUT was detectable in ocular tissues and anterior chamber after intraperitoneal injection. An intraperitoneal injection of LUT ameliorated alkali burn-elicited corneal opacity, corneal epithelial defects, collagen degradation, NV, and the infiltration of inflammatory cells. The mRNA expressions of IL-1ß, IL-6, MCP-1, vascular endothelial growth factor (VEGF)-A, and MMPs in corneal tissue were downregulated by LUT intervention. And its administration reduced the protein levels of IL-1ß, collagenases, and MMP activity. Furthermore, in vitro study showed that LUT inhibited IL-1ß-induced type I collagen degradation and the release of inflammatory cytokines and chemokines by corneal stromal fibroblasts. LUT also inhibited the IL-1ß-induced activation of TAK-1, mitogen-activated protein kinase (MAPK), c-Jun, and NF-κB signaling pathways in these cells. Our results demonstrate that LUT inhibited alkali burn-stimulated collagen breakdown and corneal inflammation, most likely by attenuating the IL-1ß signaling pathway. LUT may therefore prove to be of clinical value for treating corneal alkali burns.
Subject(s)
Burns, Chemical , Corneal Opacity , Rats , Humans , Animals , Burns, Chemical/complications , Burns, Chemical/drug therapy , Burns, Chemical/metabolism , Vascular Endothelial Growth Factor A/metabolism , Luteolin/pharmacology , Luteolin/therapeutic use , Alkalies/toxicity , Interleukin-6/metabolism , Cornea/metabolism , Cytokines/metabolism , Neovascularization, Pathologic/metabolism , Collagen Type I/metabolism , Mitogen-Activated Protein Kinases/metabolism , Corneal Opacity/metabolism , Inflammation/metabolism , Matrix Metalloproteinases/metabolismABSTRACT
The use of alkaline salt lands for crop production is hindered by a scarcity of knowledge and breeding efforts for plant alkaline tolerance. Through genome association analysis of sorghum, a naturally high-alkaline-tolerant crop, we detected a major locus, Alkaline Tolerance 1 (AT1), specifically related to alkaline-salinity sensitivity. An at1 allele with a carboxyl-terminal truncation increased sensitivity, whereas knockout of AT1 increased tolerance to alkalinity in sorghum, millet, rice, and maize. AT1 encodes an atypical G protein γ subunit that affects the phosphorylation of aquaporins to modulate the distribution of hydrogen peroxide (H2O2). These processes appear to protect plants against oxidative stress by alkali. Designing knockouts of AT1 homologs or selecting its natural nonfunctional alleles could improve crop productivity in sodic lands.
Subject(s)
Alkalies , Crops, Agricultural , GTP-Binding Protein gamma Subunits , Plant Proteins , Salt Tolerance , Sorghum , Crops, Agricultural/genetics , Crops, Agricultural/physiology , Hydrogen Peroxide/metabolism , Oryza/genetics , Oryza/physiology , Oxidative Stress/genetics , Plant Breeding , Salinity , Alkalies/analysis , Alkalies/toxicity , Sodium Bicarbonate/analysis , Sodium Bicarbonate/toxicity , Carbonates/analysis , Carbonates/toxicity , Salt Tolerance/genetics , Sorghum/genetics , Sorghum/physiology , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/physiology , Plant Proteins/genetics , Plant Proteins/physiology , Aquaporins/metabolism , Crop Production , Genetic Loci , Soil/chemistryABSTRACT
Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 µL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.
Subject(s)
Burns, Chemical , Corneal Injuries , Eye Burns , Animals , Rabbits , Corneal Stroma/pathology , Alkalies/toxicity , Burns, Chemical/pathology , Sodium Hydroxide/toxicity , Cornea/pathology , Corneal Injuries/pathology , Eye Burns/chemically induced , Eye Burns/pathology , Fibrosis , Reference StandardsABSTRACT
Saline-alkali water resources are abundant and widely distributed in China. The effective utilization of saline-alkali water resources by fishery is of great significance to enhance the aquatic product economy and restore the ecology of saline-alkali environments. Eriocheir sinensis is a saline-alkali water-suitable species. To explore its physiological response to saline-alkali stress, the hepatopancreas tissue structure, antioxidation, immunocompetence and metabolomics were investigated after 96 h of gradient saline-alkali treatment. The results confirmed the hepatopancreas damage through tissue sectioning, abnormal enzyme activity (aspartate transaminase (AST), alanine aminotransferase (ALT)) and aberrant malondialdehyde (MDA) content. The activity of superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) was significantly upregulated (p < 0.05), which was followed by a decrease trend, indicating the enhancement of antioxidant capacity in response to the stress. Strengthened immunocompetence in response to saline-alkali toxicity was shown in the gradual increase of immune enzyme activity (acid phosphatase (ACP) and alkaline phosphatase (AKP)) and the upregulated expression of immune genes (hsp 70, hsp 90, proPO and toll). Among the differential metabolites quantified by metabolomics, small peptides were significantly downregulated (p < 0.05), and acylcarnitines were obviously upregulated (p < 0.05), indicating that saline-alkali toxicity inhibited protein catabolism and stimulated the mobilization of energy reserves. Metabolic pathways enriched through the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that Eriocheir sinensis activated different mechanisms in response to various degrees of stress, such as "ABC transporters" and "purine metabolism" in response to low saline-alkali stress, while "pyrimidine metabolism" and "beta-alanine metabolism" to high saline-alkali stress.
Subject(s)
Antioxidants , Brachyura , Animals , Antioxidants/metabolism , Alkalies/toxicity , Oxidative Stress , Metabolomics , Acid Phosphatase/metabolismABSTRACT
Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.
Subject(s)
Burns, Chemical , Corneal Injuries , Corneal Neovascularization , Eye Burns , Mice , Animals , Corneal Neovascularization/drug therapy , Burns, Chemical/drug therapy , Alkalies/toxicity , Eye Burns/drug therapy , Corneal Injuries/metabolism , Cornea/metabolism , Inflammation/metabolism , Cytokines/metabolism , Disease Models, AnimalABSTRACT
PURPOSE: To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. METHODS: Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. RESULTS: NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 µM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. CONCLUSIONS: Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.
Subject(s)
Burns, Chemical , Corneal Injuries , Corneal Neovascularization , Eye Burns , Rats , Humans , Animals , Burns, Chemical/drug therapy , Vascular Endothelial Growth Factor A/metabolism , Hydrogels/pharmacology , Corneal Neovascularization/chemically induced , Corneal Neovascularization/drug therapy , Corneal Neovascularization/metabolism , Eye Burns/chemically induced , Eye Burns/drug therapy , Neovascularization, Pathologic/metabolism , Human Umbilical Vein Endothelial Cells , Alkalies/toxicity , Dexamethasone/pharmacology , Disease Models, AnimalABSTRACT
Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-ß (TGF-ß) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-ß was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.
Subject(s)
Burns, Chemical , Corneal Injuries , Eye Burns , Alkalies/toxicity , Animals , Burns, Chemical/metabolism , Corneal Injuries/pathology , Disease Models, Animal , Eosine Yellowish-(YS)/adverse effects , Eye Burns/chemically induced , Eye Burns/drug therapy , Eye Burns/pathology , Fibroblast Growth Factor 10/pharmacology , Hematoxylin/pharmacology , Hematoxylin/therapeutic use , Humans , Inflammation/drug therapy , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Nerve Growth Factor/pharmacology , Nerve Growth Factor/therapeutic use , Rabbits , Transforming Growth Factor beta/metabolism , Transforming Growth Factors/adverse effects , Wound HealingABSTRACT
Purpose: To evaluate the efficacy of losartan and prednisolone acetate in inhibiting corneal scarring fibrosis after alkali burn injury in rabbits. Methods: Sixteen New Zealand White rabbits were included. Alkali injuries were produced using 1N sodium hydroxide on a 5-mm diameter Whatman #1 filter paper for 1 minute. Four corneas in each group were treated six times per day for 1 month with 50 µL of (1) 0.8 mg/mL losartan in balanced salt solution (BSS), (2) 1% prednisolone acetate, (3) combined 0.8 mg/mL losartan and 1% prednisolone acetate, or (4) BSS. Area of opacity and total opacity were analyzed in standardized slit-lamp photos with ImageJ. Corneas in both groups were cryofixed in Optimal cutting temperature (OCT) compound at 1 month after surgery, and immunohistochemistry was performed for alpha-smooth muscle actin (α-SMA) and keratocan or transforming growth factor ß1 and collagen type IV with ImageJ quantitation. Results: Combined topical losartan and prednisolone acetate significantly decreased slit-lamp opacity area and intensity, as well as decreased stromal myofibroblast α-SMA area and intensity of staining per section and confined myofibroblasts to only the posterior stroma with repopulation of the anterior and mid-stroma with keratocan-positive keratocytes after 1 month of treatment. Corneal fibroblasts produced collagen type IV not associated with basement membranes, and this production was decreased by topical losartan. Conclusions: Combined topical losartan and prednisolone acetate decreased myofibroblast-associated fibrosis after corneal alkali burns that produced full-thickness injury, including corneal endothelial damage. Increased dosages and duration of treatment may further decrease scarring fibrosis. Translational Relevance: Topical losartan and prednisolone acetate decrease myofibroblast-mediated scarring fibrosis after corneal injury.
Subject(s)
Burns, Chemical , Corneal Diseases , Corneal Injuries , Adrenal Cortex Hormones/metabolism , Alkalies/metabolism , Alkalies/toxicity , Animals , Burns, Chemical/complications , Burns, Chemical/drug therapy , Burns, Chemical/metabolism , Cicatrix/metabolism , Cicatrix/pathology , Collagen Type IV/metabolism , Corneal Diseases/metabolism , Corneal Diseases/pathology , Corneal Injuries/complications , Corneal Injuries/drug therapy , Corneal Injuries/metabolism , Fibrosis , Losartan/metabolism , Losartan/pharmacology , Losartan/therapeutic use , Myofibroblasts/metabolism , Myofibroblasts/pathology , RabbitsABSTRACT
AIM: This study aimed to investigate the efficiency of topically applied pycnogenol (PYC) in healing the standardized alkaline corneal ulcer in diabetic and normal rats. MATERIALS AND METHODS: The corneal alkali-burn injury (CA-I) model was unilaterally developed in Wistar rats by filter paper saturated with 0.01 M of NaOH and touching the eyes for 45 s. Rats were divided into four groups: Normal control (NC), normal PYC (NPYC), diabetic control (DC), and diabetic PYC (DPYC). Both NPYC and DPYC groups were daily treated with PY eye drops three times, whereas NC and DC ones were treated with ordinary saline for six successive days. RESULTS: The wound healing of corneal epithelial was improved in the NPYC group compared to the NC group. Meanwhile, it was significantly improved (P < 0.05) in the DPYC group than in the DC group. Histological examination revealed that corneal re-epithelialization was more accomplished in the DPYC group than in the DC group. In addition, the inflammatory cells were augmented in the DC group more than those in the DPYC one. CONCLUSION: The findings obtained revealed the efficiency of PYC for enhancing the corneal re-epithelialization and reducing the inflammatory reaction post alkali burn in rats, and thus it could be beneficially valuable as a treatment for the diabetic keratopathy.
Subject(s)
Burns, Chemical , Corneal Diseases , Diabetes Mellitus, Experimental , Rodent Diseases , Alkalies/therapeutic use , Alkalies/toxicity , Animals , Burns, Chemical/drug therapy , Burns, Chemical/pathology , Burns, Chemical/veterinary , Corneal Diseases/veterinary , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/complications , Flavonoids , Plant Extracts , Rats , Rats, WistarABSTRACT
Purpose: The purpose of this study was to investigate the effects of Forkhead Domain Inhibitor-6 (FDI-6) on regulating inflammatory corneal angiogenesis and subsequent fibrosis induced by alkali burn. Methods: A corneal alkali burn model was established in Sprague Dawley rats using NaOH and the rat eyes were topically treated with FDI-6 (40 µM) or a control vehicle four times daily for 7 days. Corneal neovascularization, inflammation and epithelial defects were observed on days 1, 4, and 7 under a slit lamp microscope after corneal alkali burn. Analysis of angiogenesis-, inflammation-, and fibrosis-related indicators was conducted on day 7. Murine macrophages (RAW264.7 cells) and mouse retinal microvascular endothelial cells (MRMECs) were used to examine the effects of FDI-6 on inflammatory angiogenesis in vitro. Results: Topical delivery of FDI-6 significantly attenuated alkali burn-induced corneal inflammation, neovascularization, and fibrosis. FDI-6 suppressed the expression of angiogenic factors (vascular epidermal growth factor, CD31, matrix metalloproteinase-9, and endothelial NO synthase), fibrotic factors (α-smooth muscle actin and fibronectin), and pro-inflammatory factor interleukin-6 in alkali-injured corneas. FDI-6 downregulated the expression of monocyte chemotactic protein-1, pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-alpha), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3, and vascular endothelial growth factor in RAW264.7 cells and inhibited the proliferation, migration, and tube formation of MRMECs in vitro. Conclusions: FDI-6 can attenuate corneal neovascularization, inflammation, and fibrosis in alkali-injured corneas.
Subject(s)
Burns, Chemical , Corneal Injuries , Corneal Neovascularization , Eye Burns , Alkalies/toxicity , Animals , Burns, Chemical/complications , Burns, Chemical/drug therapy , Burns, Chemical/metabolism , Corneal Injuries/chemically induced , Corneal Injuries/complications , Corneal Injuries/drug therapy , Corneal Neovascularization/chemically induced , Corneal Neovascularization/drug therapy , Corneal Neovascularization/metabolism , Endothelial Cells/metabolism , Eye Burns/pathology , Fibrosis , Inflammation/pathology , Mice , Neovascularization, Pathologic/metabolism , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Corneal neovascularization (CoNV) in response to chemical burns is a leading cause of vision impairment. Although glutamine metabolism plays a crucial role in macrophage polarization, its regulatory effect on macrophages involved in chemical burn-induced corneal injury is not known. Here, we elucidated the connection between the reprogramming of glutamine metabolism in macrophages and the development of alkali burn-induced CoNV. Glutaminase 1 (GLS1) expression was upregulated in the mouse corneas damaged with alkali burns and was primarily located in F4/80-positive macrophages. Treatment with a selective oral GLS1 inhibitor, CB-839 (telaglenastat), significantly decreased the distribution of polarized M2 macrophages in the alkali-injured corneas and suppressed the development of CoNV. In vitro studies further demonstrated that glutamine deprivation or CB-839 treatment inhibited the proliferation, adhesion, and M2 polarization of bone marrow-derived macrophages (BMDMs) from C57BL/6J mice. CB-839 treatment markedly attenuated the secretion of proangiogenic factors, including vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from interleukin-4- (IL-4-) regulated M2 macrophages. Our findings revealed that GLS1 inhibition or glutamine deprivation prevented alkali-induced CoNV by inhibiting the infiltration and M2 polarization of macrophages. This work suggests that pharmacological GLS1 inhibition is a feasible and effective treatment strategy for chemical burn-related CoNV in humans.
Subject(s)
Corneal Neovascularization , Alkalies/toxicity , Animals , Corneal Neovascularization/chemically induced , Corneal Neovascularization/drug therapy , Glutaminase/adverse effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Many species of bacteria interact on the human skin to form a certain microbiome. Delftia acidovorans, a bacterium detected from human skin, inhibits the growth of S. epidermidis, a dominant bacterium of the human skin microbiota. Here, we show that ammonia secreted by D. acidovorans inhibits the growth of S. epidermidis by increasing the pH value of the medium. The pH value of D. acidovorans culture supernatant (CS) was higher than that of the medium without culture. The inhibitory activity of the D. acidovorans CS against the growth of S. epidermidis was decreased by neutralization with hydrochloric acid. Genes encoding enzymes related to ammonia production were found in the D. acidovorans genome. Moreover, the D. acidovorans CS contained a high concentration of ammonia. The addition of ammonia to S. epidermidis culture led to an increase in the reactive oxygen species (ROS) production and inhibited S. epidermidis growth. The addition of sodium hydroxide also led to an increase in the ROS production and inhibited S. epidermidis growth. The inhibitory activity of ammonia and sodium hydroxide against S. epidermidis growth was suppressed by malonic acid, an inhibitor of succinate dehydrogenase in the tricarboxylic acid (TCA) cycle, and N-acetyl-l-cysteine, a free radical scavenger. These findings suggest that D. acidovorans secretes ammonia and alkaline stress inhibits the growth of S. epidermidis by inducing TCA cycle-triggered ROS production.
Subject(s)
Alkalies/toxicity , Citric Acid Cycle , Reactive Oxygen Species/metabolism , Staphylococcus epidermidis/growth & development , Stress, Physiological , Ammonia/pharmacology , Delftia acidovorans/physiology , Free Radical Scavengers/pharmacology , Hydrogen-Ion Concentration , Sodium Hydroxide/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiology , Stress, Physiological/drug effectsABSTRACT
Soil salinization seriously restricts the growth and yield of soybeans. However, little information is available on the early growth stages of soybeans which are subjected to the gibberellin biosynthesis inhibitor, prohexadione-calcium (Pro-Ca). This study aimed to investigate the effects of exogenous Pro-Ca on saline-alkali stress-induced damages to photosynthesis and antioxidant defenses in soybean (Glycine max L.) seedlings. At the V3 growth stage, salt-tolerant genotype Hefeng 50 (HF50) and salt-sensitive genotype Kenfeng 16 (KF16) were subjected to 110 mmol L-1 mixed saline-alkali stress respectively, and then 100 mg L-1 Pro-Ca was sprayed on the leaves. Our results showed that saline-alkali stress accelerated the degradation of thylakoids, inhibited chlorophyll synthesis, reduced shoot dry weight, electron transfer rate (ETR), and peroxidase (POD) activity, the concentration of ascorbic acid (AsA) and soluble sugar, but enhanced the concentration of proline, hydrogen peroxide (H2O2) and the rate of superoxide radical (O2â-) generation. Additionally, saline-alkali stress induced a lower decrease of the net photosynthetic rate (Pn), potential activity of PSII (Fv/F0), and maximum quantum yield of PSII (Fv/Fm) in salt-tolerant HF50 than in salt-sensitive KF16. Nevertheless, foliar spraying of exogenous Pro-Ca increased the chlorophyll content, Pn, Fv/F0, and Fv/Fm. These results were more prominent when Pro-Ca was applied to KF16 under saline-alkali conditions. Furthermore, exogenous application of Pro-Ca retarded the degradation of thylakoids, increased the ETR and the accumulation of AsA, soluble sugar, and proline, activated the activities of superoxide dismutase (SOD), catalase (CAT), and POD, and decreased the concentration of malondialdehyde (MDA), electrolyte leakage (EL), O2â-, and H2O2. These results indicated that Pro-Ca could effectively protect soybean seedlings against damage from saline-alkali stress by regulating seedling phenotype, photosynthetic apparatus, antioxidant defense, and osmoregulation.
Subject(s)
Alkalies/toxicity , Antioxidants/metabolism , Glycine max/drug effects , Ketoglutaric Acids/pharmacology , Photosynthesis/drug effects , Chlorophyll/metabolism , Plant Leaves/drug effects , Plant Leaves/metabolism , Seedlings/drug effects , Seedlings/growth & development , Seedlings/metabolism , Glycine max/growth & development , Glycine max/metabolism , Stress, Physiological/drug effects , Thylakoids/metabolismABSTRACT
The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.